This invention relates to methods of cell disruption and plasmid extraction in the field of recombinant DNA technology. Specifically, it relates to mechanical methods of rupturing cells to release intact plasmids cloned within the cells.
In the field of recombinant DNA technology, plasmid expression vectors are routinely employed to express foreign proteins. A number of recombinant proteins, including recombinant human insulin (HUMULIN(copyright), Lilly), recombinant human erythropoietin (EPOGEN(copyright), Amgen), recombinant tissue plasminogen activator (ACTIVASE(copyright), Genentech), and recombinant xcex1 interferon (ROFERON(copyright), Roche), are now available for human pharmaceutical use, and commercial scale methods have been developed for recovery and purification of recombinant proteins from cell culture and/or microbial fermentation. For most of recombinant proteins produced in mammalian cell culture and for some recombinant proteins produced in microbes but secreted into the culture medium, cell disruption is not required for the recovery of these products. When cell disruption is required to release intracellular recombinant products from microbes, mechanical cell rupture methods are frequently used in such large recombinant protein recovery processes.
More recently, it has been shown that plasmid DNA may be useful as a non-viral nucleic acid delivery vehicle for clinical applications. (See, e.g., Wang et al., Proc. Nat""l Acad. Sci. USA 90:4156-4160 (1993); Ulmer et al., Science 259:1745-1749 (1993)). For such applications, which include gene therapy and genetic immunization, the plasmids themselves rather than the expressed proteins are the desired therapeutic product. Accordingly, there is a need for pharmaceutically acceptable large scale processes for recovery of intact plasmid DNA. For a number of reasons, mechanical cell disruption methods are preferred to chemical or enzymatical cell disruption methods if the yields are comparable.
Bacterial plasmids are double-stranded closed circular DNA molecules that range in size from about 1 kb to more than 200 kb. They are found in a variety of bacterial species, where they serve as accessary genetic units that replicate and are inherited independently of the bacterial chromosome. Plasmids can be produced via bacterial fermentation and recovered by cell disruption and plasmid recovery operations. Fermentation technology to produce plasmids is relatively well understood, and a number of laboratory scale methods useful for bacterial cultures ranging in size from 1 mL to 1 L have been developed to purify plasmid DNA from bacteria. (See Sambrook, Fritsch and Maniatis, Section 1.21, xe2x80x9cExtraction and Purification of Plasmid DNAxe2x80x9d, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989).) These methods involve the growth of the bacterial culture and replication of plasmid; harvesting and lysis of the bacteria; isolation and purification of plasmid DNA.
Following growth of the bacterial culture, bacteria are normally recovered by centrifugation and lysed by one of a number of methods, including treatment with enzymes, nonionic or ionic detergents, organic solvents, alkali, or heat. The choice of lytic method is influenced by factors such as the size of the plasmid, the strain of bacteria used, and methods to be used subsequently to purify the plasmid DNA. Although well suited for small scale processes, enzymatic or chemical lysis are rather expensive. Chemical lysis also limits the choice of the downstream processing techniques used subsequently to purify the plasmid DNA. Enzymatic lysis frequently uses animal-derived enzymes such as lysozyme, which maybe accompanied with animal virus. Significant efforts to validate the removal of any possible viral contaminations are needed for this situation.
The currently published laboratory methods are in general unsatisfactory for large scale plasmid purification processes. Laboratory methods for isolation and purification of plasmids from bacterial culture frequently use dangerous organic solvents and chemicals such as cesium chloride and ethidium bromide, which are in general unacceptable for human pharmaceutical use. The few studies related to large scale plasmid recovery that have been reported, (See Chandra, G. et al., Analytical Biochemistry 203:169-172 (1992); Chakrabarti, A. et al., Biotechnology and Applied Biochemistry 16:211-215 (1992)), use chemical methods of cell lysis, i.e., alkaline-SDS lysis. However, SDS can cause significant problems in downstream purification.
In general laboratory scale plasmid purification methods were developed for gene cloning purposes, in which case, bacterial genomic DNA and tRNA or rRNA impurities as well as damaged plasmid are relatively unimportant. In contrast, plasmid DNA for pharmaceutical use must meet extremely high standards of identity and purity; this necessitates stringent limits on nucleic acid and protein impurities. Non-plasmid DNA, RNA, as well as plasmid DNA fragments may need to be removed in the downstream purification process. As intact plasmid DNA is ordinarily separable from both the larger intact host cell genomic DNA and from smaller cellular RNAs and DNAs on the basis of size and chemistry, it is important to avoid shearing either the plasmid DNA or the genomic DNA of the host organism. Linearized plasmid DNA and genomic DNA fragments similar in size to the intact plasmid product may be particular difficult to remove.
Bacterial plasmids for clinical applications typically contain large segments of product DNA (mammalian DNA for gene therapy applications or pathogen DNA for genetic immunization), as well as the expression vectors themselves, which contain the genes for selection in bacteria, the sequences for replication in bacteria, and the regulatory elements for expression in mammalian cells. Such plasmid molecules tend to be large, on the order of 106-107 Daltons (5-20 kb), which is approximately two to three orders of magnitude larger than typical recombinant protein products (e.g., human growth hormone at 104 Dalton). Large plasmids (greater than 10 kb size) are particularly susceptible to damage, especially by physical forces that might be necessary to release the plasmid from the interior of the cell.
Although mechanical methods of cell disruption would be more economical and easier to carry out, and therefore preferred to enzymatic or chemical cell disruption methods for large scale processes, it is recognized that mechanical methods may damage DNA at the same time as the cells are broken. (Wheelwright, S. M., Protein Putification:Design and Scale up of Downstream Processing, Oxford University Press (1991), in Chapter 6: Cell Disruption, p. 63. This presents a significant potential problem for pharmaceutical use, where intact, functional plasmid DNA is required and supercoiled plasmid DNA is preferred. Plasmid DNA which has been xe2x80x9cnickedxe2x80x9d but not cut through both strands, loses it supercoiled configuration and becomes xe2x80x9crelaxedxe2x80x9d circular DNA. Supercoiled plasmid DNA, which is smaller and more compact than relaxed closed circular plasmid DNA and less vulnerable to enzymatic degradation, expresses better than either relaxed circular or linear DNA. Although supercoiled plasmid DNA is preferred, both supercoiled and relaxed circular plasmid DNA are likely to express the gene of interest and are considered xe2x80x9cintactxe2x80x9d plasmid DNA. Although plasmid linearized using a selected restriction enzyme may constitute a functional expression unit, mechanical forces are likely to cut or break plasmid in a random manner. Randomly linearized plasmid DNA and broken or fragmented plasmid DNA are considered damaged and are likely to be ineffective or nonfunctional for pharmaceutical purposes. Not only is such damaged plasmid DNA ineffective, it will probably need to be removed in downstream processing to achieve a higher standard of purity. To permit recovery of intact plasmid DNA, processing conditions must be very mild, particularly with respect to shear forces. Although enzymatic and chemical lysis methods tend to involve little or no shear force, it presents other problems as discussed previously. Various methods of cell lysis, including certain mechanical methods, are available when it is not necessary to obtain intact plasmid DNA; however, the development of pharmaceutically acceptable large scale procedures for mechanical cell disruption yielding intact plasmid DNA presents a substantial challenge for biochemical engineers.
Several processing methods for the disruption of bacterial cells are commonly used to release intracellular protein products. These include: 1) sonication (Neppiras, E. A. and Hughes, D. E., xe2x80x9cSome experiments on the disintegration of yeast by high intensity soundxe2x80x9d, Biotechnology and Bioengineering, 6:247-270 (1964)); 2) homogenization (Kula, M.-R. and Schutte, H., xe2x80x9cPurification of proteins and the disruption of microbial cellsxe2x80x9d, Biotechnology Process 3(1):31-42 (1987)); 3) microfluidization (Sauer, T., Robinson, C. W., and Glick, B. R., xe2x80x9cDisruption of native and recombinant Escherichia coli in a high pressure homogenizerxe2x80x9d, Biotechnology and Bioengineering, 33:1330-1342 (1989)); Agerkvist, I., and Enfors, S.-O., xe2x80x9cCharacterization of E. coli cell disintegrates from a bead mill and high pressure homogenizersxe2x80x9d, Biotechnology and Bioengineering 36:1083-1089 (1990); 4) bead milling (Kula and Schutte, supra; Limon-Lason, J., Hoare, M., Osborn, C. B., Doyle, D. J., and Dunnill, P., xe2x80x9cReactor properties of a high speed bead mill for microbial cell rupturexe2x80x9d, Biotechnology and Bioengineering 21(5):745-774 (1979); Marffy, F. and Kula, M. R., xe2x80x9cEnzyme yields from cells of brewers yeast disrupted by treatment in a horizontal disintegratorxe2x80x9d, Biotechnology and Bioengineering 16:632-634 (1974)); and more recently 5) nebulization. The effectiveness of these processes has been studied to a limited extent when intracellular proteins were the product of interest or when cell disruption was the only goal (Marffy and Kula, supra; Woodrow, J. R. and Quirk, A. V., xe2x80x9cEvaluation of the potential of a bead mill for the release of intracellular bacterial enzymesxe2x80x9d, Enzyme and Microbial Technology 4(6):385-389 (1982); and Schutte, H., Kroner, K. H., Hustedt, H., and Kula M. R., xe2x80x9cExperiences with a 20 liter industrial bead mill for the disruption of microorganismsxe2x80x9d, Enzyme and Microbial Technology 5(2):143-148 (1983)). The findings show that all of the above mentioned methods are effective for bacterial cell disruption and that the disruption depends on the conditions of residence time, pressure, agitation rate, and other equipment variables as appropriate for the particular device. The major difference between the different methods is related to the size of the cell fragments generated, with some of the methods disrupting cells with less overall destruction of the cell envelope. This is significant since the size of the fragments generated has an important impact on further downstream processing when these particles are removed from the lysate or, minimally, separated from other subcellular species. (Agerkvist and Enfors, supra, and Mosqueira, F. G., Higgins, J. J., Dunnill, P. and Lilly, M. D., xe2x80x9cCharacteristics of mechanically disrupted baker""s yeast in relation to its separation in industrial centrifugesxe2x80x9d, Biotechnology and Bioengineering 23:335-343 (1981)).
In addition to the effect of processing on the size of the cell fragments generated, some studies have shown that the severity of the cell disruption conditions can have an impact on the yield of active protein recovered in the process. (Marffy and Kula, supra.) Generally speaking, the amount of active protein found in the liquid phase (i.e., outside the cells) increases in proportion to the fraction of cells disrupted early in the disruption process, but then decreases with further processing. This behavior fits a model of the process in which the protein is released from the cells by disruption by a first order process (Dakubu, S., xe2x80x9cCell Inactivation by Ultrasoundxe2x80x9d, Biotechnology and Bioengineering 18:465-471 (1979); Marffy and Kula, supra; Limon-Lason, et al., supra), then is deactivated by the effects of the disruption process. There seems to be general agreement that protein deactivation is caused by shearing at the molecular level or by thermal denaturation caused by local overheating of the suspension fluid (Marffy and Kula, supra; Chisti and Moo-Young, 1986, xe2x80x9cDisruption of Microbial Cells for Intracellular Productsxe2x80x9d, Enzyme and Microbial Technology 8:194-204 (1986)). However, it is also clear that different proteins behave quite differently, i.e., some are easily deactivated and suffer severely from overprocessing, while others are more stable and are persistent in the product. Determination of acceptable processing techniques is primarily empirical and involves systematic manipulation of the severity of the disruption, the residence time, and the number of passes or amount of reprocessing of the cell suspension. Optimization of recovery processes generally involves the assumption that the disruption and deactivation processes can be modeled as sequential first order reactions. This suggests certain disruption motifs, such as avoiding well stirred reactors which tend to minimize intermediate product formation, and optimization of reactor residence time and pseudo rate constants. Little work has been done to confirm the behavior of actual systems.
To the extent that the effects of mechanical cell disruption on cellular DNA have been evaluated, DNA fragmentation has in general been considered desirable in that it reduces the viscosity of the solution, thereby making protein recovery easier. (Agerkvist and Enfors, supra). In contrast to protein isolation studies, few if any studies have been conducted on cell disruption processes when DNA is the final product to be recovered. Although the general problem is similar to that with protein release, in that the processes required to disrupt the cells also tend to destroy the product molecule, because the DNA tends to be a larger molecule, it is much more sensitive to shear generated in most disruption methods than are proteins. For this reason, the destruction process can be very rapid, and the yields of released but intact nucleic acid molecules can be very low. Because of the potential for destruction of the product, the standard disruption methods must all be reevaluated when the product molecule is DNA or RNA, particularly when it is necessary to recover intact plasmid DNA.
We evaluated five different mechanical disruption processes to determine their potential as cell disruption methods for DNA (plasmid) products. A model host-plasmid system was grown for cell paste. The cell paste was isolated then resuspended in a TE disruption buffer and the suspension was processed through different bench or pilot scale cell disruption equipment under conditions suggested by the manufacturers, reported in publications, as well as developed by the inventor of this patent. The intact cells and cell debris were separated from soluble molecules by high speed centrifugation in microfuge tubes, and the amount of intact plasmid DNA remaining in the cells, released into the liquid phase, and destroyed in the disruption process were measured by quantitative gel electrophoresis with the aid of image analysis equipment. The findings show that most common disruption methods result in almost complete destruction of released plasmid DNA and consequently very low plasmid yields. However, two methods appear to be relatively mild in terms of shearing the plasmids and could be used for high yield recovery of intact plasmid from bacterial cells, preferably E. coli. 
One embodiment of the invention relates to a mechanical method for disruption of plasmid-containing bacterial cells and release of intact plasmid DNA which can then be isolated. The method comprises the steps of first passing liquid suspension of plasmid-containing bacterial cells between one and three times through an impinging-jet homogenizer with a single interaction chamber at an operating pressure of about 750 to 4,000 psi, preferably about 1,000 to 3,000 psi, more preferably about 2,000 psi, whereby the bacterial cells are disrupted and intact plasmid DNA is released. The disrupted bacterial cell debris is then separated from the liquid containing intact plasmid DNA. The plasmid may then be further isolated and purified. Another embodiment of the invention relates to a mechanical method for disruption of plasmid-containing bacterial cells and release of intact plasmid DNA. The method comprises the steps of first passing liquid containing plasmid-containing bacterial cells through a bead mill containing beads of about 0.1 mm to about 1 mm in diameter, at an agitation speed of about 1,000 to 2,500 rpm. Because, such lower-speed agitation disrupts cells with minimal damage to plasmid contained therein, either batch mode, single-pass processing or multiple pass processing can be used so long as the total processing times are similar. With multiple pass processing, the liquid is passed through the bead mill at least two times, preferably four to eight times, more preferably between five and six times, for a residence time in the bead mill of about 0.5 to about 3 minutes per pass, whereby bacterial cells are disrupted and intact plasmid DNA is released. If a single batch mode, using a single pass operation is used, the liquid suspension of plasmid-containing bacterial cells is processed in the bead mill for at least three minutes, preferably between about five and thirty five minutes, more preferably between about ten and twenty minutes, whereby bacterial cells are disrupted and intact plasmid DNA is released. The disrupted bacterial cell debris is then separated from the liquid containing intact plasmid DNA. The plasmid may then be further isolated and purified.